Shanghai Key Laboratory of Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 197# Ruijin Er Road, Shanghai, 200025, People's Republic of China.
J Gastroenterol. 2013 Sep;48(9):1023-33. doi: 10.1007/s00535-012-0733-6. Epub 2013 Jan 22.
MicroRNAs can promote or suppress the evolution of malignant behaviors by regulating multiple targets. We aimed to determine the expression of miR-301a recently screened in gastric cancer, to investigate the biological effects of miR-301a and to identify the specific miR-301a target gene.
Quantitative real-time RT-PCR was used to test miR-301a expression. Functional effects were explored by a water-soluble tetrazolium salt assay, a colony formation assay in soft agar, a migration assay, an invasion assay and cytometry used to determine apoptosis and cell cycle. Nude mice were inoculated subcutaneously by retrovirus-mediated stably expressed SGC-7901 cells. The target gene was determined by bioinformatic algorithms, dual luciferase reporter assay and Western blot.
Firstly, we found that miR-301a was significantly upregulated both in cells and tissues of gastric cancer. The expression level of miR-301a was inversely correlated with tumor differentiation of gastric cancer tissues. Secondly, miR-301a promoted cell growth, soft agar clonogenicity, migration, invasion, and decreased cell apoptosis induced by cisplatin in vitro, while blockage of miR-301a reduced the percentage of G2/M phase cells via flow cytometry in gastric cancer cells. Ectopic expression of miR-301a enhanced the subcutaneous tumorigenesis in vivo. Finally, miR-301a directly downregulated RUNX3 expression post-transcriptionally in gastric cancer.
Our results demonstrate that miR-301a plays important roles in the development of gastric cancer.
microRNAs 可以通过调节多个靶标促进或抑制恶性行为的演变。我们旨在确定最近在胃癌中筛选出的 miR-301a 的表达，研究 miR-301a 的生物学效应，并确定特定的 miR-301a 靶基因。
使用实时定量 RT-PCR 测试 miR-301a 的表达。通过水溶性四唑盐 assay、软琼脂集落形成 assay、迁移 assay、侵袭 assay 和流式细胞术检测细胞凋亡和细胞周期来探索功能效应。通过逆转录病毒介导的稳定表达 SGC-7901 细胞，在裸鼠中皮下接种。通过生物信息学算法、双荧光素酶报告基因 assay 和 Western blot 确定靶基因。
首先，我们发现 miR-301a 在胃癌细胞和组织中均显著上调。miR-301a 的表达水平与胃癌组织的肿瘤分化程度呈负相关。其次，miR-301a 促进了细胞生长、软琼脂集落形成、迁移、侵袭，并降低了顺铂诱导的细胞凋亡，而通过流式细胞术检测到，miR-301a 阻断可使胃癌细胞 G2/M 期细胞比例减少。过表达 miR-301a 增强了体内皮下肿瘤的发生。最后，miR-301a 直接下调胃癌中 RUNX3 的转录后表达。
我们的研究结果表明，miR-301a 在胃癌的发展中起着重要作用。
